scholarly journals Architecture of the yeast origin recognition complex bound to origins of DNA replication.

1997 ◽  
Vol 17 (12) ◽  
pp. 7159-7168 ◽  
Author(s):  
D G Lee ◽  
S P Bell
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Origin Recognition Complex ◽  
Binding Assays ◽  
Chromosomal Replication ◽  
Origins Of Replication ◽  
Eukaryotic Dna ◽  
Level Of Analysis ◽  
Origin Recognition

In many organisms, the replication of DNA requires the binding of a protein called the initiator to DNA sites referred to as origins of replication. Analyses of multiple initiator proteins bound to their cognate origins have provided important insights into the mechanism by which DNA replication is initiated. To extend this level of analysis to the study of eukaryotic chromosomal replication, we have investigated the architecture of the Saccharomyces cerevisiae origin recognition complex (ORC) bound to yeast origins of replication. Determination of DNA residues important for ORC-origin association indicated that ORC interacts preferentially with one strand of the ARS1 origin of replication. DNA binding assays using ORC complexes lacking one of the six subunits demonstrated that the DNA binding domain of ORC requires the coordinate action of five of the six ORC subunits. Protein-DNA cross-linking studies suggested that recognition of origin sequences is mediated primarily by two different groups of ORC subunits that make sequence-specific contacts with two distinct regions of the DNA. Implications of these findings for ORC function and the mechanism of initiation of eukaryotic DNA replication are discussed.

scholarly journals Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA

2020 ◽  
Vol 48 (19) ◽  
pp. 11146-11161
Author(s):  
Naining Xu ◽  
Yingying You ◽  
Changdong Liu ◽  
Maxim Balasov ◽  
Lee Tung Lun ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Solution Structure ◽  
Origin Recognition Complex ◽  
Structural Basis ◽  
Dna Replication Origin ◽  
Dna Alterations ◽  
Replication Initiator ◽  
Binding Subunit ◽  
Origin Recognition

Abstract The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

scholarly journals Role of the Orc6 Protein in Origin Recognition Complex-Dependent DNA Binding and Replication in Drosophila melanogaster

2007 ◽  
Vol 27 (8) ◽  
pp. 3143-3153 ◽  
Author(s):  
Maxim Balasov ◽  
Richard P. H. Huijbregts ◽  
Igor Chesnokov
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Dna Binding ◽  
Origin Recognition Complex ◽  
Dominant Negative ◽  
The Core ◽  
Culture Cells ◽  
Replication Domain ◽  
Origin Recognition

ABSTRACT The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.

scholarly journals Two subunits of human ORC are dispensable for DNA replication and proliferation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Etsuko Shibata ◽  
Manjari Kiran ◽  
Yoshiyuki Shibata ◽  
Samarendra Singh ◽  
Shashi Kiran ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cell Lines ◽  
Human Cell ◽  
Origin Recognition Complex ◽  
Human Cell Lines ◽  
Atpase Subunit ◽  
Origins Of Replication ◽  
A Subunit ◽  
Dependent Mechanism ◽  
Origin Recognition

The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1. The ORC1 or ORC2-depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication

scholarly journals Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro

2000 ◽  
Vol 14 (13) ◽  
pp. 1631-1641 ◽  
Author(s):  
Tohru Mizushima ◽  
Naoko Takahashi ◽  
Bruce Stillman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Origin Recognition Complex ◽  
Binding Activity ◽  
Chromosomal Dna ◽  
Dna Binding Activity ◽  
Dna Binding Specificity ◽  
Dna Replication Origins ◽  
Origin Recognition

An interaction between the origin recognition complex (ORC) and Cdc6p is the first and a key step in the initiation of chromosomal DNA replication. We describe the assembly of an origin-dependent complex containing ORC and Cdc6p from Saccharomyces cerevisiae. Cdc6p increases the DNA binding specificity of ORC by inhibiting non-specific DNA binding of ORC. Cdc6p induces a concomitant change in the conformation of ORC and mutations in the Cdc6p Walker A and Walker B motifs, or ATP-γ-S inhibited these activities of Cdc6p. These data suggest that Cdc6p modifies ORC function at DNA replication origins. On the basis of these results in yeast, we propose that Cdc6p may be an essential determinant of origin specificity in metazoan species.

scholarly journals Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites

2001 ◽  
Vol 21 (8) ◽  
pp. 2790-2801 ◽  
Author(s):  
James F. Theis ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Binding Site ◽  
Binding Sites ◽  
Origin Recognition Complex ◽  
Replication Origins ◽  
Discrete Site ◽  
Origin Function ◽  
Single Binding Site ◽  
Origin Recognition

ABSTRACT While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins inSaccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

scholarly journals Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

2001 ◽  
Vol 21 (17) ◽  
pp. 5767-5777 ◽  
Author(s):  
Amit Vas ◽  
Winnie Mok ◽  
Janet Leatherwood
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Origin Recognition Complex ◽  
Safety Net ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Phosphorylation Sites ◽  
Cdc2 Kinase ◽  
Origin Recognition ◽  
Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

scholarly journals The Origin Recognition Complex Interacts with a Subset of Metabolic Genes Tightly Linked to Origins of Replication

PLoS Genetics ◽  
2009 ◽  
Vol 5 (12) ◽  
pp. e1000755 ◽  
Author(s):  
Erika Shor ◽  
Christopher L. Warren ◽  
Joshua Tietjen ◽  
Zhonggang Hou ◽  
Ulrika Müller ◽  
...  
Keyword(s):  
Origin Recognition Complex ◽  
Metabolic Genes ◽  
Origins Of Replication ◽  
Origin Recognition

scholarly journals Dendrite development

2005 ◽  
Vol 170 (4) ◽  
pp. 517-519 ◽  
Author(s):  
Michael D. Ehlers
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
The Core ◽  
Dendrite Development ◽  
Origin Recognition

Neurons extend elaborate dendrites studded with spines. Unexpectedly, this cellular sculpting is regulated by the origin recognition complex—the core machinery for initiating DNA replication.

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